The lipocalins are a diverse family of small and robust, secretory proteins which serve for the transport or storage of poorly soluble or chemically sensitive vitamins, hormones, and metabolites (such as retinoids, fatty acids, cholesterols, prostaglandins, biliverdins, pheromones, tastants, and odorants) in many organisms (Åkerström et al. Eds. (2006), Lipocalins, Landes Bioscience, Georgetown, Tex.; Pervaiz, S., and Brew, K. (1987) FASEB J. 1, 209-214). Although they have, in the past, been classified primarily as transport proteins, it is now clear that the lipocalins fulfill a variety of physiological functions. These include roles in retinol transport, olfaction, pheromone signaling, and the synthesis of prostaglandins. The lipocalins have also been implicated in the regulation of the immune response and the mediation of cell homoeostasis (reviewed, for example, in Flower, D. R. (1996) Biochem. J. 318, 1-14 and Flower, D. R. et al. (2000) Biochim. Biophys. Acta 1482, 9-24).
The lipocalins share unusually low levels of overall sequence conservation, often with sequence identities of less than 20%. In strong contrast, their overall folding pattern is highly conserved. The central part of the lipocalin structure consists of a single eight-stranded anti-parallel β-sheet closed back on itself to form a continuously hydrogen-bonded β-barrel. One end of the barrel is sterically blocked by the N-terminal peptide segment that runs across its bottom as well as three peptide loops connecting the β-strands. The other end of the β-barrel is open to the solvent and encompasses a target-binding site, which is formed by four peptide loops. It is this diversity of the loops in the otherwise rigid lipocalin scaffold that gives rise to a variety of different binding modes each capable of accommodating targets of different size, shape, and chemical character (reviewed, e.g., in Flower, D. R. (1996), supra; Flower, D. R. et al. (2000), supra, or Skerra, A. (2000) Biochim. Biophys. Acta 1482, 337-350).
Among the 10-12 members of the lipocalin family that are found in the human body, human neutrophil gelatinase-associated lipocalin (hNGAL) (Kjeldsen et al. (2000) Biochim. Biophys. Acta 1482, 272-283)—also known as lipocalin 2 (Lcn2) or, more recently, dubbed siderocalin (Goetz et al. (2002) Mol. Cell. 10, 1033-1043)—plays a role in the innate immune defence against bacterial infections by scavenging Fe3+ ions bound to certain bacterial siderophores.
Such siderophores are highly potent iron chelators which are secreted by pathogenic bacteria in response to limiting iron concentrations (Schaible & Kaufmann (2004). Nat. Rev. Microbial. 2, 946-953), as they happen in the human body fluids, to allow iron uptake by specialized bacterial import systems (Braun & Braun (2002) Curr. Opin. Microbial. 5, 194-201; Fischbach et al. (2006) Nat. Chem. Biol. 2, 132-138). It seems that neutrophils release hNGAL at sites of infection as an antimicrobial strategy. Indeed, the physiological relevance of hNGAL has been demonstrated in corresponding knock-out mice, where this lipocalin was shown to be essential in limiting the spreading of bacteria that rely on enterobactin-mediated iron import (Flo et al. (2004) Nature 432, 917-921)
hNGAL (also termed Lcn2, SWISS-PROT Data Bank Accession Number P80188) is a 178 amino acid glycoprotein with strong binding activity towards the catecholate-type siderophore Fe3+-enterobactin (or enterochelin), which is characteristic for Escherichia coli (Raymond et al. (2003) Proc. Natl. Acad. Sci. USA 100, 3584-8). hNGAL is an abundant human plasma protein, whose normal concentration is around 80 μg/L and can increase up to ten-fold upon bacterial infections (Xu and Venge (2000) Biochim. Biophys. Acta 1482, 298-307), and its single N-linked glycosylation site is dispensable for folding (Coles et al. (1999) J. Mol. Biol. 289, 139-157). Compared with other lipocalins, hNGAL exhibits an unusually large pocket. Therein, a cluster of positively charged side chains confers extraordinary affinity for the negatively charged ferric siderophore, with a dissociation constant (KD) of 0.4 nM (Goetz et al., supra), thus allowing effective competition with the bacterial uptake system. Ligand recognition by hNGAL is rather specific as this lipocalin also forms stable complexes with the chemically related bacillibactin from Bacillus anthracis (Abergel et al. (2006) Proc. Natl. Acad. Sci. USA 103, 18499-18503) and with carboxymycobactins from Mycobacterium tuberculosis (Holmes et al. (2005) Structure 13, 29-41), a siderophore type of similar size and shape. However, it does not bind petrobactin, the siderophore that is crucial for virulence of B. anthracis (Abergel et al., supra), or C-glycosylated enterobactin analogues such as the salmochelins produced by Salmonella spp. and Klebsiella pneumoniae (Fischbach et al., supra). Animal homologs to human Lcn2 are rat α2-microglobulin-related protein (A2m; SWISS-PROT Data Bank Accession Number P31052) and mouse 24p3/uterocalin (24p3; SWISS-PROT Data Bank Accession Number P11672).
Proteins that selectively bind to their corresponding targets by way of non-covalent interaction play a crucial role as reagents in biotechnology, medicine, bioanalytics as well as in the biological and life sciences in general. Antibodies, i.e. immunoglobulins, are a prominent example of this class of proteins. Despite the manifold needs for such proteins in conjunction with recognition, binding and/or separation of ligands/targets, almost exclusively immunoglobulins are currently used. The application of other proteins with defined ligand-binding characteristics, for example the lectins, has remained restricted to special cases.
Rather recently, members of the lipocalin family have become subject of research concerning proteins having defined ligand-binding properties. The PCT publication WO 99/16873 discloses polypeptides of the lipocalin family with mutated amino acid positions in the region of the four peptide loops, which are arranged at the end of the cylindrical β-barrel structure encompassing the binding pocket, and which correspond to those segments in the linear polypeptide sequence comprising the amino acid positions 28 to 45, 58 to 69, 86 to 99, and 114 to 129 of the bilin-binding protein of Pieris brassicae. 
The PCT publication WO 00/75308 discloses muteins of the bilin-binding protein, which specifically bind digoxigenin, whereas the International Patent Applications WO 03/029463 and WO 03/029471 relate to muteins of the human neutrophil gelatinase-associated lipocalin (hNGAL) and apolipoprotein D, respectively. In order to further improve and fine tune ligand affinity, specificity as well as folding stability of a lipocalin variant various approaches using different members of the lipocalin family have been proposed (Skerra, A. (2001) Rev. Mol. Biotechnol. 74, 257-275; Schlehuber, S., and Skerra, A. (2002) Biophys. Chem. 96, 213-228), such as the replacement of additional amino acid residues. The PCT publication WO 2006/56464 discloses muteins of human neutrophil gelatinase-associated lipocalin with binding affinity for CTLA-4 in the low nanomolar range.
The PCT publication WO 2005/19256 discloses muteins of tear lipocalin with at least one binding site for different or the same target ligand and provides a method for the generation of such muteins of human tear lipocalin. According to this PCT application, certain amino acid stretches within the primary sequence of tear lipocalin, in particular the loop regions comprising amino acids 7-14, 24-36, 41-49, 53-66, 69-77, 79-84, 87-98, and 103-110 of mature human tear lipocalin, are subjected to mutagenesis in order to generate muteins with binding affinities. The resulting muteins have binding affinities for the selected ligand (KD) in the nanomolar range.
The lipocalin muteins disclosed in the above references are selected to preferentially bind large, proteinaceous target molecules and not small molecules. Thus, despite the progress made in this field, it would be desirable to have hNGAL muteins that are specifically adapted to bind small molecules with high binding affinity, for example in the nanomolar range. Such muteins would further improve the suitability of muteins of hNGAL in diagnostic and therapeutic applications.